CN105891463A - Beta-HCG quantitative detection kit based on nanometer magnetic particle time resolution fluorescence - Google Patents
Beta-HCG quantitative detection kit based on nanometer magnetic particle time resolution fluorescence Download PDFInfo
- Publication number
- CN105891463A CN105891463A CN201410857408.0A CN201410857408A CN105891463A CN 105891463 A CN105891463 A CN 105891463A CN 201410857408 A CN201410857408 A CN 201410857408A CN 105891463 A CN105891463 A CN 105891463A
- Authority
- CN
- China
- Prior art keywords
- hcg
- magnetic particle
- kit
- monoclonal antibody
- antibody
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Abstract
The invention discloses a beta-HCG quantitative detection kit based on nanometer magnetic particle time resolution fluorescence. A calibrator is involved, magnetic particle suspension liquid of an anti-beta-HCG monoclonal antibody and a europium-marked anti-beta-HCG monoclonal antibody are combined, and a buffer solution, a washing solution, an enhancement solution and a reaction tube are analyzed. Magnetic particle-beta-HCG-europium-marked antibody two-antibody sandwich compound and the free europium-marked beta-HCG monoclonal antibody are separated, the enhancement solution is added, and the absorbance value is measured. Magnetic particles are dispersed in liquid, the reaction bonding area is enlarged, matter to be detected is enriched, reaction time is shortened, and meanwhile the kit has the advantages that marker preparation is easy and convenient, storage time is long, radioactive contamination is avoided, the standard curve range is wide and interferences of sample autofluorescence are avoided. According to the kit, sensitivity is high, accuracy is good, reaction time is short, manual operation can be achieved, automation can also be achieved, errors can be greatly reduced, and the reliability of a detection result is improved.
Description
Technical field:
The invention belongs to bioanalytical chemistry, biological technical field, specifically, relate to a kind of based on nano magnetic particulate time-resolved fluorescence
β-HCG immue quantitative detection reagent box.
Background technology:
Human chorionic gonadotrophin (human chorionic gonadotropin, HCG) is to be produced by the plamoditrophoblast of Chorionic villi of placenta
A kind of glycoprotein, bis-subunits of α and β be combined into by ionic bond and hydrophobic bond.Alpha subunit is that tethelin is common, it
Identical with the alpha subunit structure of FSH, LH and TSH, and β subunit is peculiar by HCG, in immunocompetence can with FSH, LH and
TSH etc. distinguish.Free β human chorionic gonadotrophin (free β-HCG) refers to the β subunit of the HCG of unbound state, HCG in blood
Major part exists with full molecular forms, and freeβfreing-HCG accounts for the 1～5% of total HCG.
β-HCG is significant to First Trimester diagnosis, sees the diagnosis and the course of disease with the disease such as pregnancy related disorder, trophoblastic tumor
Examine certain values.During gestation, in blood, β-HCG is within 6～8 days after becoming pregnant, to start to produce, and within 50～80 days, reaches peak, the most steadily declines, directly
To within pregnant about 18 weeks, being stable at finite concentration.
Down syndrome also known as mongolism, trisomy 21 syndrome, be cause retarded common disease because of one of.At present by examining in pregnant morning, mid-term
Survey PAPP-A, β-HCG, AFP and ultrasound diagnosis in blood of pregnant women, can detect that the Down syndrome of about 90% comes.A part of pregnant have Tang Shi combine
The pregnant woman of simulator sickness fetus, its serum procollagen Ⅲ level is straightforward rising, and second trimester of pregnancy, Down syndrome pregnancy serum β-HCG concentration ratio was the most pregnant
Woman is the highest more than 2 times.
β-HCG detection at present uses EIA enzyme immunoassay (Enzymatic immunoassay, EIA), chemiluminescence immune assay mostly
(chemiluminescence immunoassay, CLIA) and time resolved fluoro-immunoassay (time-resolved
Fluoroimmunoassay, TRFIA) etc..It is low to there is sensitivity in EIA, and the range of linearity is narrow, is difficult to realize the methodology defects such as automation.CLIA
There is the advantages such as the harm of "dead" radiation, highly sensitive, detection range of linearity width, good stability, automaticity height and range of application width,
It is widely used clinically.But there is also that luminescence process is short, sample can not duplicate detection, background higher and easy by surrounding material interference etc.
Shortcoming.And current domestic use major part be expensive import reagent, such as Roche, Abbott, Beckman etc..
Magnetic particle immunoassay technology is to utilize a certain size Magnetic solid phases particulate of synthesis of polymer material to make carrier, with physical absorption or chemistry
The method of coupling is by high specific and the antibody of high-affinity or antigen coated on magnetic particle.Magnetic particle has following compared with traditional microwell plate
Feature: surface area is bigger, can be in conjunction with more protein molecular；Can be connected with probe molecule by covalent bond, more firm than the physical absorption of microwell plate
Gu；It is the solid phase carrier of a kind of flowing, makes reaction rapider.In real work, testing sample often contains more impurity component, quickly divides
It is one of difficult point of clinical examination worker from purification of target determinand.The magnetic particle specific binding target determinand of energy, can under additional magnetic fields
Directed movement, makes target determinand be separated, concentration and purifying, has that separating rate is fast, efficiency is high, favorable repeatability, simple to operate, no
Affect by the feature such as small protein biological character and function of separation.
TRFIA technology is after radiommunoassay, a new milestone of label development, it has also become in clinical ultramicron biochemical investigation
One analysis means the most promising.TRFIA, using lanthanide series as label, is most commonly used that europium element.The fluorescence lifetime of lanthanide series
Long, Strokes displacement is big, non-specific signals can be reduced to negligible degree, reach high signal to noise ratio.Europium label has system
Standby easy, store time length, standard curve range width, "dead" pollution, quite varied etc. unexcellent by the interference of sample natural fluorescence and range of application
Point, overcomes the instability of enzyme marker, chemiluminescence is only capable of once luminous and easily by environmental disturbances, the shortcoming such as non-immediate mark of electrochemical luminescence.
But, the many employings of producer TRFIA based on microwell plate technology the most both at home and abroad.Owing to the solid-liquid phase reaction area of microwell plate is little, during the reaction of needs
Between longer, microwell plate coated antibody or antigen are to be coated by physical absorption, coated antibody or antigen standardization is difficult and less stable so that detection
Result precision is poor.TRFIA technology automaticity based on microwell plate is low, the most semi-automatic, or pre-treatment is plus TRFIA detector
Quasi-fully-automated synthesis.
The chemiluminescence immune assay being currently based on immunity magnetic particle is used widely in clinic, but glimmering based on immunity magnetic particle time resolution
β-HCG the detection of light immunoassay there is no document report.Magnetic particle time-resolved fluorescence technology combines magnetic particle immunoassay technology and divides with the time
Distinguish the feature of fluoroimmunoassay, it is possible to reduce the sample size needed for reaction, accelerate the reaction time, easily realize automation and sample detects immediately.
Summary of the invention
It is an object of the invention to provide a kind of β-HCG immue quantitative detection reagent box based on nano magnetic particulate time-resolved fluorescence.
The methodology basis of the present invention is double-antibody sandwich immune response: be connected formation solid-phase complex by particulate with β-HCG antibody, i.e.
Immunity magnetic particle, with the determined antigen β-HCG in immunity magnetic particle capture sample, then is attached to be captured with the β-HCG antibody of europium mark
On β-HCG, ultimately form the compound of immunity magnetic particle-β-HCG antigen-europium labeling antibody.Free Europium label is removed in washing, adds and strengthens liquid,
Under the exciting of ultraviolet light, launch strong fluorescence, measure its fluorescence intensity, fluorescence intensity and the β-HCG in testing sample with time identifier
Concentration is directly proportional, and reference standard curve i.e. can determine that the concentration of β-HCG in sample.
β-HCG the immue quantitative detection reagent box based on nano magnetic particulate time-resolved fluorescence of the present invention includes: 1) connect anti-β-HCG antibody
Magnetic particle；2) europium marks anti-β-HCG antibody；
Alternatively, any one or the youngster's kind during mentioned reagent box also includes following reagent and/or device: 3) calibration object；4) analysis buffer；
5) cleaning solution (25x)；6) liquid is strengthened；7) reaction tube.
Alternatively, the β-HCG immue quantitative detection reagent box based on nano magnetic particulate time-resolved fluorescence of the present invention may include that 1) calibration object；
2) magnetic particle of anti-β-HCG antibody is connected；3) europium marks anti-β-HCG antibody.4) analysis buffer；5) cleaning solution (25x)；6) liquid is strengthened；
7) reaction tube.
The preparation process of the magnetic particle of above-mentioned connection anti-β-HCG monoclonal antibody is: prepared by magnetic particle → magnetic particle activation → with anti-β-HCG
Monoclonal antibody connects, alternatively, and the step of washing after also including antibody connection → close → preservation.Coupling Gao Te can be prepared with reference to above step
The immune magnetic particle of the anti-β-HCG monoclonal antibody of the opposite sex.
Above-mentioned magnetic particle has superparamagnetism, and its diameter is preferably 10～50nm, and the content of the carboxyl-reactive group that every gram of magnetic particle is carried is not
Less than 0.4mmol；The described anti-β preferred purity of-HCG monoclonal antibody is more than or equal to 90%, dilutes titer more than 1: 100 ten thousand；Described idol
Connection agent is carbodiimide.
The above-mentioned magnetic particle preparation method containing carboxyl-reactive group is: by FeCl3·6H2O and FeCl2.4H2O adds with mol ratio 2: 1
In distilled water, dissolved with vigorous agitation；Add 0.5M ammoniacal liquor in a nitrogen environment, hatch 45min for 65 DEG C；Then distilled water washing, abandons supernatant,
60 DEG C of drying magnetic particles；By magnetic particle ultrasonic disperse at 10% (w/v) PEG8000 solution, add the absolute ethyl alcohol of 10% (v/v)；At nitrogen
Adding crosslinking agent N under compression ring border, N '-methylene-bisacrylamide, 30min is hatched in 61 DEG C of stirrings；Add 3% (v/v) benzoyl peroxide,
The styrene of 50% (v/v), the acrylic acid of 25% (v/v)；Distilled water cyclic washing after reaction 8-10h, removes uncoated Fe3O4Magnetic, vacuum is done
Surface must be arrived after dry and be associated with the magnetic particle of carboxyl.
Above-mentioned europium marks the preparation process of anti-β-HCG antibody: select the β-HCG monoclonal antibody for another antigen site to carry out Eu3+
Mark, antibody and Eu3+The mass ratio of label can be 1: 10 to 10: 1, preferably 1: 1,2: 1,3: 1,4: 1,5: 1, most preferably quality
Ratio is 5: 1.Labeling process is: by anti-β-HCG monoclonal antibody with N1-(P mono-isothiocyanatobenzyl)-diethylenetriamineteacidcetic acidcetic europium sodium with 1: 5
Ratio mixing, 4 DEG C stand overnight, and then use Sephacryl S-200 splitter, respectively with containing 0.85%NaCl (w/v) and
The free Eu of 50mmol/L Tris-HCl wash-out3+Label is combined Eu with antibody3+Label.
The preparation process of above-mentioned calibration object is: with containing 2g/L BSA and 1g/L NaN3Tris-HCl buffer solution, its concentration is preferably 50
Mmol/L, pH are preferably 7.8, β-HCG psma ligand is made 0,5,20,50,500, the calibration of the series concentration of 5000mlU/mL molten
Liquid.Dispense and be stored in 2-8 DEG C；
Above-mentioned analysis buffer formula is: Tris-HCl (50mmol/L), PEG6000 (1.5%, w/v), BSA (0.5%, w/v),
Proclin300 (0.1%, v/v), ox 1gG (0.02%, w/v), Tween20 (0.1%, v/v), NaCl (0.84%, w/v), pH7.8's
Buffer solution；
Above-mentioned cleaning solution (25 ×) formula is: Tris-HCl (1.25mmol/L), Tween20 (2.5%, v/v), NaCl (21%, w/v),
The buffer solution of pH7.8.
Above-mentioned enhancing formula of liquid is: by β-naphthoyltrifluoroacetone (β-NTA, 15 μm ol/L), trioctylphosphine oxide (TOPO,
50 μm ol/L), Triton X-100 (0.1%, v/v), acetic acid (0.6%, v/v) forms, and adjusts pH with the Potassium Hydrogen Phthalate of 6.8nmol/L
It is 3.0～3.2；
The material of above-mentioned reaction tube is transparent polystyrene, polyethylene, polypropylene or glass.
The assay method of the present invention is: every reaction tube adds use the analysis buffer of 50 μ l with 1: the 25 immune magnetic particle diluted, and is subsequently adding
β-HCG calibration object or sample 50 μ l, add the Eu with analysis buffer 1: 50 dilution3+Labelled antibody 100 μ l, room temperature concussion hatch 30min,
Using magnetic separation technique will be coated immune magnetic particle and the supernatant separating, washing of β-HCG, last every hole adds 200 μ l and strengthens liquid, shakes 5min
After on time-resolved fluorescence detector measure fluorescent value.
Compared with existing β-HCG detection kit, kit of the present invention has the advantage that
(1) this kit combines magnetic separation technique and TRFIA technology, has both had highly sensitive, storage time length, the line of TRFIA technology
Property the advantage such as wide ranges, "dead" pollution, the most fully spread also by the immunity enrichment of magnetic particle and magnetic particle and increase and combine table
The effect of enlarged areas, is greatly shortened the reaction time, improves detection sensitivity；
(2) connect magnetic particle and antibody by chemical group orientation, add antibody binding strength, decrease antibody consumption, also improve detection
Precision；
(3) this technology both can carry out manual operations, also can realize automation.When for automatic analyzer, can measure instant by single sample
Detection, also can batch detection, overcoming conventional microporous board-like TRFIA technology can only the shortcoming of batch detection.
Accompanying drawing explanation
Fig. 1 is the measurement result comparison diagram that the kit of the present invention and the particulate chemical luminescence reagent kit of Abbott measure β-HCG, wherein horizontal
Coordinate is the β-HCG value of kit measurement of the present invention, and ordinate is the particulate chemical luminescence reagent kit measured value of Abbott, two kinds of methods
Linear correlation equation is y=0.989x-0.618, coefficient R2=0.979.
Detailed description of the invention
Embodiment 1: the preparation of the kit of the present invention:
(1) preparation of β-HCG calibration object:
It is 50mmol/L by concentration, containing 2g/L BSA and 1g/L NaN3Tris-HCl buffer solution, by β-HCG antigen (purchased from China inspection
Test calibrating research institute) be configured to 0,5,20,50,500, the calibration solution of the series concentration of 5000mlU/mL.
(2) preparation of the magnetic particle suspension of anti-β-HCG monoclonal antibody is combined:
By FeCl3·6H2O and FeCl2.4H2O joins in distilled water with mol ratio 2: 1, dissolved with vigorous agitation；Add 0.5M ammonia in a nitrogen environment
Water, hatches 45min for 65 DEG C；Then a large amount of distilled water washings, abandon supernatant, 60 DEG C of drying magnetic particles；By magnetic particle ultrasonic disperse at 10% (w/v)
PEG8000 solution, adds the absolute ethyl alcohol of 10% (v/v)；Addition crosslinking agent N in a nitrogen environment, N '-methylene-bisacrylamide, 61 DEG C
30min is hatched in stirring；Add the benzoyl peroxide of 3% (v/v), the styrene of 50% (v/v), the acrylic acid of 25% (v/v)；Reaction 8-10h
Rear distilled water cyclic washing, must arrive surface and be associated with the magnetic particle of carboxyl after vacuum drying.Then magnetic particle (is purchased with anti-β-HCG monoclonal antibody
From Fitzgerald company, magnetic particle and antibody mass ratio 1: 10) in the presence of coupling agent carbodiimide (1g/L), room temperature reaction 4～6 hours,
Reaction is washed and is closed after terminating, and adjusts pH and concentration with buffer solution, is 7.8 to pH, and concentration is 0.5mg/mL, and 2～8 DEG C of refrigerations are standby
With.
(3) preparation of the anti-β-HCG antibody of europium mark:
Another strain is added 0.5ml 50mmol/L for anti-β-HCG monoclonal antibody 1mg (purchased from Fitzgerald company) of different epitopes
Na2CO3Buffer solution, is centrifuged 5min with Millipore company with the centrifuge tube 10000rpm of the G-50 of filter membrane, repeats washing 5 times, falls
Leaving the heart and collect antibody, centrifugal purification antibody, at 200 microliters, is joined 0.2mg Eu by fixing fabric structure3+Label is (purchased from PerKinElmer
Company) in, 4 DEG C stand overnight.Then Sephacryl S-200 splitter is used, respectively with containing 0.85%NaCl and 50mmol/L
The free Eu of Tris-HCl wash-out3+Label is combined Eu with antibody3+Label.Collect efflux (1ml/ pipe) by pipe, and measure absorbance (A280nm),
After merging peak pipe and with 1.0ml/ bottle vacuum freeze drying 2～8 DEG C of preservations.
(4) analysis buffer formula is: Tris-HCl (50mmol/L), PEG6000 (1.5%, w/v), BSA (0.5%, w/v), Proclin300
(0.1%, v/v), ox 1gG (0.02%, w/v), Tween20 (0.1%, v/v), NaCl (0.84%, w/v), the buffer solution of pH7.8；
(5) cleaning solution (25x) formula is: Tris-HCl (1.25mmol/L), Tween20 (2.5%, v/v), NaCl (21%, w/v), pH7.8's
Buffer solution.
(6) strengthening formula of liquid is: by β-naphthoyltrifluoroacetone (β-NTA, 15 μm ol/L), trioctylphosphine oxide (TOPO, 50 μm ol/L), Triton
X-100 (0.1%, v/v), acetic acid (0.6%, v/v) forms, and adjusting pH with the Potassium Hydrogen Phthalate of 6.8nmol/L is 3.0～3.2.
Embodiment 2: the performance indications inspection of reagent of the present invention:
(1) kit calibration object is analyzed measuring with the national serial standards of respective concentration simultaneously, uses double-log Model fitting, it is desirable to two
Bar dose-response curve is not deviating significantly for parallel (t checks, | t | ＜ 2.447)；With company standard product as reference substance, use double-log Model fitting,
The mean value of the measured value of kit calibration object and sign value ratio should be in the range of 0.9～1.1.The linearly dependent coefficient of dose-response curve
(r)=0.996.
(2) sensitivity: by null value normative reference product duplicate measurements 8 times, calculate its fluorescent value and standard deviation.The gained of 2 times of standard deviations is added with this average
Fluorescent value substitutes into calibration curve and calculates its concentration value, and this value is its sensitivity, and the sensitivity of this reagent analysis is 20mlU/ml after measured.
(3) range of linearity: be measured after antigen diluent becomes variable concentrations, recording the calibration curve range of linearity is 5～5000mlU/ml, for dense
The degree sample more than 5000mIU/mL should be diluted measuring.
(4) precision (CV%): the respectively sample (expection concentration is respectively as follows: 2,15,5000mlU/mL) of the high, medium and low concentration of replication,
The variation within batch coefficient recording kit of the present invention is 2.2%～6.2%, and interassay coefficient of variation is 3.7%～8.1%.
Table 1: batch interior, betweenrun precision test result (n=20)
(5) specific
| The cross reaction factor | Concentration | Measured value |
| Interstitialcellstimulating hormone (ICSH) (LH) | 1000ng/mL | ＜ 2ng/mL |
| Follicle-stimulating hormone (FSH) (FSH) | 1000ng/mL | ＜ 2ng/mL |
| Thyrotropic hormone (TSH) | 1000μlU/mL | ＜ 2ng/mL |
(6) stability: opening latter 37 DEG C and place 7 days, measured value should meet above-mentioned requirements.
The using method of the kit of embodiment 3 present invention
(1) sample collection: collection venous blood 2～3ml, in anticoagulant heparin pipe, is centrifuged 3～5min separated plasmas with 3500～4000rpm.Blood plasma sample
Product can be stablized more than 7 days 2～8 DEG C of preservations, if needing long-term preservation, please-20 DEG C of preservations.
(2) preparation of reagent:
1. kit to be checked is balanced 30 minutes under room temperature (18～25 DEG C).
2. cleaning solution is prepared: washing lotion deionization 25 times dilution will be concentrated, be and make cleaning solution.
3. preparation Europium label working solution: use first 1 hour by every bottle of label 1mL deionized water dissolving, by analysis buffer with 1: 50
Dilution is as paving labeling antibody working solution again.
4. immunity magnetic particle: need concussion to suspend before use and mix.
(3) operating procedure:
1. every reaction tube adds the dilution magnetic particle of 50 μ l, is subsequently adding 50 μ l calibration object or samples, adds 100 μ l europium labelled antibodies, room
Temperature concussion incubation reaction 30min.
2. reaction tube is placed in magnetic separation frame upper 2 minute, makes magnetic particle aggegation.
3. rinse 4 times with cleaning solution, each cleaning solution consumption 300 μ l, need to stand 30 seconds after adding cleaning solution every time, make magnetic particle aggegation again
Inhale again and abandon supernatant.
4. every hole adds enhancing liquid 200 μ l, and concussion is upper machine mensuration after hatching 5 minutes.
The clinical comparison experiment of 4 kits of embodiment
The kit of invention has been carried out clinical comparison experiment, by the most right for the particulate chemical luminescence reagent kit of this kit and Abbott
β-HCG the concentration of 154 example clinical samples is measured.With this kits value as abscissa, Abbott's kit measurement value is ordinate,
Two groups of measurement results carry out regression analysis, and gained linear correlation equation is: y=0.989x-0.618, coefficient R2=0.979.Linear regression
Coefficient is 0.989 close to 1, it is seen that kit prepared by this method and hospital's measured value have preferable uniformity.With SPSS13.0 statistical analysis software pair
Coefficient correlation carries out t inspection (inspection level α=0.05), P ＜ 0.001.β-HCG the value that visible two kinds of methods measure is closely related, and examination is described
The diagnosis capability of agent box is relatively strong, can promote clinical practice.
In order to determine the clinical reference value of this kit, the plasma sample to the 215 nogestational health objects of example, use this kit to carry out β-HCG
Concentration measures, and result shows that the term of reference (reference interval) of this kit is: 0-5mlU/ml.
Claims (10)
1. a β-HCG immue quantitative detection reagent box based on nano magnetic particulate time-resolved fluorescence, it is characterised in that including: combine anti-β-HCG Dan Ke
The magnetic particle suspension of grand antibody, the anti-β-HCG monoclonal antibody of europium mark, calibration object, analysis buffer, cleaning solution, enhancing liquid.
2. kit as claimed in claim 1, it is characterised in that the preparation process of described calibration object is: with containing 2g/L BSA and 1g/L NaN3
Tris-HCl buffer solution, its Tris-HCl concentration be 50mmol/L, pH be 7.8, β-HCG psma ligand is made 0,5,20,50,
500, the calibration solution of the series concentration of 5000mlU/mL.
3. kit as claimed in claim 1, it is characterised in that the preparation method of the magnetic particle suspension of described combination anti-β-HCG monoclonal antibody
As follows: to make the magnetic particle containing carboxyl-reactive group with anti-β-HCG monoclonal antibody in the presence of coupling agent, room temperature reaction 4～6 hours,
Reaction is washed and is closed after terminating.
Kit the most according to claim 3, it is characterised in that described magnetic particle has superparamagnetism, its diameter is preferably 10～50nm,
The content of the carboxyl-reactive group carried on every gram of magnetic particle is not less than 0.4mmol；The described anti-β preferred purity of-HCG monoclonal antibody is more than
Equal to 90%, dilute titer more than 1: 100 ten thousand；Described coupling agent is carbodiimide.
Kit the most according to claim 3, it is characterised in that the described magnetic particle preparation method containing carboxyl-reactive group is: will
FeCl3·6H2O and FeCl2.4H2O joins in distilled water with mol ratio 2: 1, dissolved with vigorous agitation；Add 0.5M ammonia in a nitrogen environment
Water, hatches 45min for 65 DEG C；Then distilled water washing, abandons supernatant, 60 DEG C of drying magnetic particles；By magnetic particle ultrasonic disperse 10%
(w/v) PEG8000 solution, adds the absolute ethyl alcohol of 10% (v/v)；Addition crosslinking agent N in a nitrogen environment, N '-methylene-bisacrylamide,
30min is hatched in 61 DEG C of stirrings；Add the benzoyl peroxide of 3% (v/v), the styrene of 50% (v/v), the acrylic acid of 25% (v/v)；Reaction
Distilled water cyclic washing after 8-10h, removes uncoated Fe3O4Magnetic, must arrive surface and be associated with the magnetic particle of carboxyl after vacuum drying.
6. kit as claimed in claim 1, it is characterised in that described europium marks the preparation method of anti-β-HCG antibody and is: select anti-β-HCG
Monoclonal antibody carries out Eu3+Mark, labeling process is as follows: by anti-β-HCG monoclonal antibody and N1-(P mono-isothiocyanatobenzyl)-diethyl
Triamine tetraacethyl europium sodium mixes with the ratio of 1: 5, and 4 DEG C stand overnight, and then uses Sephacryl S-200 splitter, uses respectively
Containing 0.85%NaCl (w/v) and 50mmol/L Tris-The free Eu of HCl wash-out3+Label is combined Eu with antibody3+Label.
7. kit as claimed in claim 1, it is characterised in that described analysis buffer formula is: Tns-HCl (50mmol/L), PEG6000
(1.5%, w/v), BSA (0.5%, w/v), Proclin300 (0.1%, w/v), ox IgG (0.02%, w/v), Tween20 (0.1%,
V/v), NaCl (0.84%, w/v), the buffer solution of pH7.8.
8. kit as claimed in claim 1, it is characterised in that described cleaning solution formula is: Tris-HCl (1.25mmol/L), Tween20
(2.5%, v/v), NaCl (21%, w/v), the buffer solution of pH7.8.
9. kit as claimed in claim 1, it is characterised in that described enhancing formula of liquid includes: by β-naphthoyltrifluoroacetone (β-NTA, 15
μm ol/L), trioctylphosphine oxide (TOPO, 50 μm ol/L), Triton X-100 (0.1%, v/v), acetic acid (0.6%, v/v) forms, and uses
It is 3.0～3.2 that the Potassium Hydrogen Phthalate of 6.8nmol/L adjusts pH.
10. the preparation method of kit, it comprises the following steps:
1) preparation of calibration object；
2) preparation of the magnetic particle earnestly supernatant liquid of anti-β-HCG monoclonal antibody is combined；
3) europium marks anti-β-HCG monoclonal antibody；
Preferably, also include preparing analysis buffer, cleaning solution, enhancing liquid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410857408.0A CN105891463A (en) | 2014-12-31 | 2014-12-31 | Beta-HCG quantitative detection kit based on nanometer magnetic particle time resolution fluorescence |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410857408.0A CN105891463A (en) | 2014-12-31 | 2014-12-31 | Beta-HCG quantitative detection kit based on nanometer magnetic particle time resolution fluorescence |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105891463A true CN105891463A (en) | 2016-08-24 |
Family
ID=56700538
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410857408.0A Pending CN105891463A (en) | 2014-12-31 | 2014-12-31 | Beta-HCG quantitative detection kit based on nanometer magnetic particle time resolution fluorescence |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105891463A (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107132347A (en) * | 2016-11-25 | 2017-09-05 | 南方医科大学 | A kind of time-resolved fluorescence detection kit of 5 fluorouracil blood concentrations of real-time monitoring |
| CN108181287A (en) * | 2018-02-09 | 2018-06-19 | 广州源起健康科技有限公司 | A kind of magnetic bead time-resolved fluoroimmunoassay quantitatively detects H-FABP kits |
| CN108333357A (en) * | 2018-02-09 | 2018-07-27 | 广州源起健康科技有限公司 | A kind of magnetic bead time-resolved fluoroimmunoassay quantitatively detects CK-MB kits |
| CN109298178A (en) * | 2018-11-06 | 2019-02-01 | 江苏美克医学技术有限公司 | Cardiac myosin binding protein C(cMyBP-C based on immunomagnetic beads) time-resolved fluoroimmunoassay kit |
| CN109298177A (en) * | 2018-11-06 | 2019-02-01 | 江苏美克医学技术有限公司 | Time-resolved fluorescence immunoassay method based on Magneto separate |
| CN111948408A (en) * | 2020-08-31 | 2020-11-17 | 浙江盛域医疗技术有限公司 | Time-resolved fluoroimmunoassay kit for detecting TM and application thereof |
| CN111948407A (en) * | 2020-08-31 | 2020-11-17 | 浙江盛域医疗技术有限公司 | Time-resolved fluorescence immunoassay kit for detecting PIC and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101614741A (en) * | 2008-06-25 | 2009-12-30 | 上海新波生物技术有限公司 | Diagnostic kit for quantitatively detecting hepatitis B virus surface antigen |
| CN101819210A (en) * | 2009-12-08 | 2010-09-01 | 广州市丰华生物工程有限公司 | Beta-human chorionic gonadotrophin test kit (time-resolved fluoroimmunoassay) for prenatal screening and preparation method thereof |
| CN102507947A (en) * | 2011-11-11 | 2012-06-20 | 南方医科大学 | CEA TRFIA (time-resolved fluoroimmunoassay) kit based on IMB (immunomagnetic beads) |
| CN103308677A (en) * | 2013-06-14 | 2013-09-18 | 南京医科大学第二附属医院 | Chemiluminescent immune quantitative detection kit of estradiol nanometer magnetic particles and preparation method thereof |
| CN104034906A (en) * | 2014-06-24 | 2014-09-10 | 广西博士海意信息科技有限公司 | Time-resolved immunofluorescence analysis method for beta-HCG (Human Chorionic Gonadotropin) detection and kit |
| CN104181310A (en) * | 2014-07-30 | 2014-12-03 | 美艾利尔（上海）诊断产品有限公司 | Time resolution immunity fluorescence kit for detecting content of beta-hCG as well as preparation method and application thereof |
-
2014
- 2014-12-31 CN CN201410857408.0A patent/CN105891463A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101614741A (en) * | 2008-06-25 | 2009-12-30 | 上海新波生物技术有限公司 | Diagnostic kit for quantitatively detecting hepatitis B virus surface antigen |
| CN101819210A (en) * | 2009-12-08 | 2010-09-01 | 广州市丰华生物工程有限公司 | Beta-human chorionic gonadotrophin test kit (time-resolved fluoroimmunoassay) for prenatal screening and preparation method thereof |
| CN102507947A (en) * | 2011-11-11 | 2012-06-20 | 南方医科大学 | CEA TRFIA (time-resolved fluoroimmunoassay) kit based on IMB (immunomagnetic beads) |
| CN103308677A (en) * | 2013-06-14 | 2013-09-18 | 南京医科大学第二附属医院 | Chemiluminescent immune quantitative detection kit of estradiol nanometer magnetic particles and preparation method thereof |
| CN104034906A (en) * | 2014-06-24 | 2014-09-10 | 广西博士海意信息科技有限公司 | Time-resolved immunofluorescence analysis method for beta-HCG (Human Chorionic Gonadotropin) detection and kit |
| CN104181310A (en) * | 2014-07-30 | 2014-12-03 | 美艾利尔（上海）诊断产品有限公司 | Time resolution immunity fluorescence kit for detecting content of beta-hCG as well as preparation method and application thereof |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107132347A (en) * | 2016-11-25 | 2017-09-05 | 南方医科大学 | A kind of time-resolved fluorescence detection kit of 5 fluorouracil blood concentrations of real-time monitoring |
| CN108181287A (en) * | 2018-02-09 | 2018-06-19 | 广州源起健康科技有限公司 | A kind of magnetic bead time-resolved fluoroimmunoassay quantitatively detects H-FABP kits |
| CN108333357A (en) * | 2018-02-09 | 2018-07-27 | 广州源起健康科技有限公司 | A kind of magnetic bead time-resolved fluoroimmunoassay quantitatively detects CK-MB kits |
| CN109298178A (en) * | 2018-11-06 | 2019-02-01 | 江苏美克医学技术有限公司 | Cardiac myosin binding protein C(cMyBP-C based on immunomagnetic beads) time-resolved fluoroimmunoassay kit |
| CN109298177A (en) * | 2018-11-06 | 2019-02-01 | 江苏美克医学技术有限公司 | Time-resolved fluorescence immunoassay method based on Magneto separate |
| CN111948408A (en) * | 2020-08-31 | 2020-11-17 | 浙江盛域医疗技术有限公司 | Time-resolved fluoroimmunoassay kit for detecting TM and application thereof |
| CN111948407A (en) * | 2020-08-31 | 2020-11-17 | 浙江盛域医疗技术有限公司 | Time-resolved fluorescence immunoassay kit for detecting PIC and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105891463A (en) | Beta-HCG quantitative detection kit based on nanometer magnetic particle time resolution fluorescence | |
| WO2018120620A1 (en) | Fluorescence immunochromatographic detection card and preparation method therefor and use thereof | |
| CN102359958B (en) | Kit and method for detecting procalcitonin | |
| WO2016127318A1 (en) | Cardiac troponin i ultra-sensitive detection reagent kit, and ultra-sensitive detection method therefor | |
| CN102998467B (en) | β human chorionic gonadotrophin magnetic microparticle chemiluminescence immune quantitative detection reagent box and preparation method thereof | |
| CN102507947B (en) | CEA TRFIA (time-resolved fluoroimmunoassay) kit based on IMB (immunomagnetic beads) | |
| CN103698535B (en) | Platelet-activating factor acetylhydro-lase immue quantitative detection reagent box and preparation, method of operating | |
| CN104634980A (en) | Cardiac troponin I (cTn I) hypersensitive detection kit and hypersensitive detection method | |
| CN101819206B (en) | AFP (Alpha-Fetoprotein) testing kit (time-resolved fluoroimmunoassay) for prenatal screening and preparation method thereof | |
| CN105759061A (en) | Human serum adiponectin detection kit based on micro-particle chemiluminescence immune assay | |
| CN104698184A (en) | Kit for detecting carbohydrate antigen as well as detection method and application thereof | |
| CN102690863B (en) | ATP (adenosine triphosphate) testing based lymphocyte proliferation activity analysis reagent kit and preparation and application thereof | |
| CN103293323B (en) | A kind of interstitialcellstimulating hormone (ICSH) nano magnetic particulate chemistry electrochemiluminescent immunoassay immue quantitative detection reagent box and preparation method thereof | |
| CN103278651A (en) | Kit for chemiluminescence immunity quantitative detection of MYO (myohaemoglobinnano) nano magnetic particle and preparation method of kit | |
| CN102901812A (en) | Magnetic particle chemiluminescence immunoassay kit and assay method for human thyroglobulin antibodies (TGAb) | |
| CN103076458A (en) | Dissociate human chorionic gonadotrophin beta-subunit magnetic particle chemiluminescence quantitative assay kit and its preparation method | |
| CN104422772A (en) | Time resolution immunochromatographic test strip for quantitatively detecting pepsinogen I as well as preparation method of time resolution immunochromatographic test strip | |
| CN102721804A (en) | Electrical chemiluminescence immunoassay based on gold magnetic particles | |
| CN107796803A (en) | A kind of the magnetic microparticle chemiluminescence detection kit and preparation method of free β human chorionic gonadotrophins | |
| CN103344634A (en) | Chemiluminescence immune assay kit and method for magnetic particle of human alpha fetoprotein (AFP) | |
| CN101750502A (en) | TRFIA for synchronously detecting AFP and AFP-IgM and reagent kit thereof | |
| CN101377515A (en) | Thyrotropin chemiluminescence immune analysis determination reagent kit and preparing method thereof | |
| CN109211867A (en) | A kind of micro-fluidic fluorescence immunoassay chip of rapid quantitative detection BNP | |
| CN106959372A (en) | Serum amyloid A protein and the two-in-one measure kit of C reactive proteins and preparation method | |
| CN111175491A (en) | sBCMA magnetic particle chemiluminescence immunoassay kit and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20160824 |
|
| WD01 | Invention patent application deemed withdrawn after publication |